Automated biological sample collection system and methods

ABSTRACT

An automated biological sample collection system and methods are disclosed. The system comprises an incubator, an applicator and a sampler. The incubator is configured to control the environment of a plurality of eggs, the applicator is configured to deliver exogenous material to the plurality of eggs, at least one sampler is configured to collect samples from the plurality of eggs, invasively or non-invasively and a system controller is operable to drive the various components of the system automatically according to assay specifications and configuration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part of U.S. patent applicationSer. No. 13/017,011, filed Jan. 30, 2011, which claims the benefit ofpriority from U.S. Provisional Patent Application No. 61/300,071, filedFeb. 1, 2010. The contents of the above-referenced applications areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The embodiments disclosed herein relate to an automated samplecollection system and method. In particular a system is disclosed forperforming assays on fertilized and embryonated eggs.

BACKGROUND OF THE INVENTION

The embryonic chick has been used as an in vivo model for theinvestigation of a number of biological systems for over a century. Itis noted for its simplicity, availability, immune-deficient properties,and for the highly vascularized structure of the ChorioallantoicMembrane (CAM).

Many in vivo systems, particularly model systems for studying humantissues and organs, require transplanting an examined graft into animmune compromised animal, in order to avoid graft rejection. Suchanimals, for example, Severe Combined Immune Deficient (SCID) mice, areexpensive, sickly and hard to maintain.

The avian egg provides a low cost and readily available alternative toin vivo testing upon sentient animals. The avian egg has been used ingrowth of viruses for vaccine generation, angiogenesis assays,teratogenicity testing, tumor cells and the like.

Such assays may be performed on the embryos, or on microbes, cells andtissues grafted, injected or applied to fertilized avian eggs. Forexample, the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) assay isa well-known method for screening the irritancy potential of topicallyapplied compositions such as cosmetics.

However, such procedures are generally performed manually, are laborintensive and are not practical on the large scale. Furthermore, becausethere is human intervention when handling the eggs as well as in thesampling and analysis of the assays, these procedures are not easilyrepeatable. It is therefore difficult to produce large numbers ofidentical eggs and assays to be used for statistical analysis.

It will be appreciated, therefore, that there is a need for an automatedprocess for producing biotechnological assays. The system and methoddescribed herein address this need.

SUMMARY OF THE INVENTION

Embodiments described herein disclose a biological sample collectionsystem. The system comprises at least one incubator, configured tocontrol the environment of at least one egg; at least one applicator,configured to deliver exogenous material to the at least one egg; and atleast one sampler, configured to collect samples from the at least oneegg. Typically, the egg comprises a fertilized avian egg. Optionally,the at least one egg comprises a chimeric system.

Embodiments described herein disclose a method for use in an automatedsample collection system for collecting a set of biological samples fromat least one fertilized egg, the system comprises: at least oneincubator configured to control the environment of the at least onefertilized egg; a shell removal apparatus configured to remove at leasta section of the shell providing access to a chorioallantroic membrane(CAM) of the at least one fertilized egg; at least one applicatorconfigured to deliver exogenous material to the fertilized egg; at leastone sampler configured to collect samples from the at least onefertilized egg: and at least one controller unit,

the method for operating the sample collection system such that theassociated process of collecting the samples is performed in an improvedmanner, the method comprising the steps:

-   -   configuring, by the controller unit, the automated sample        collection system according to at least one automated setup        parameter;    -   pricking, by the shell removal apparatus, the chorioallantroic        membrane (CAM) of the at least one fertilized egg;    -   delivering, by the at least one applicator, at least one        exogenous material to the at least one fertilized egg using at        least one delivery mechanism associated with the at least one        sampler;    -   incubating, by the at least one incubator, the at least one        fertilized egg for a period of incubation; and    -   harvesting, by the at least one sampler, at least one sample        from the at least one fertilized egg.

The step of pricking the chorioallantroic membrane (CAM) of the at leastone fertilized egg, comprises: removing at least a part of the shell ofthe at least one fertilized egg at the widest side comprising an airpocket; and exposing at least a part of the chorioallantoic membrane(CAM).

Where appropriate, the step of pricking, further comprises: placing atube into the chorioallantoic membrane (CAM).

The step of delivering at least one exogenous material comprisesengrafting, by at least one delivery mechanism, a population of cells tosaid at least one fertilized egg.

Where appropriate, the step of delivering at least one exogenousmaterial, comprises injecting, by at least one delivery mechanism, atleast one exogenous material into a designated blood vessel of at leastone fertilized egg.

The step of injecting at least one exogenous material, comprises:directing the injection into the designated blood vessel in thedirection of blood flow.

Where required, the method may further comprise: determining thedirection of blood flow in the designated blood vessel.

Where required, the method may further comprise: selecting thedesignated blood vessel having a known direction of blood flow.

As appropriate, the step of determining the direction of blood flow inthe designated blood vessel comprises: using a flow-detector todetermine the blood flow direction of the designated blood vessel.

As appropriate, the step of determining the direction of blood flow inthe designated blood vessel, comprises: using image processing techniqueconfigured to determine the direction of blood flow according to theshape of the designated blood vessel.

As appropriate, the step of directing the injection into the designatedblood vessel in the direction of blood flow comprises: identifying anumbilical vein connecting the chorioallantoic membrane (CAM) and anembryo of the at least one fertilized egg, and injecting the exogenousmaterial into the umbilical vein in a direction from the chorioallantoicmembrane (CAM) to the embryo.

As appropriate, the step of injecting at least one exogenous materialcomprises: using the at least one delivery mechanism comprising a 30gauge needle.

Variously, the at least one exogenous material is selected from at leastone of a group consisting of: cells, tumor cells, bacteria, viruses,chemicals, drugs, skin explants and external modulators.

As appropriate, the period of incubation is selected to be sufficient toallow engraftment of the population of cells to the chorioallantoicmembrane (CAM) of the at least one fertilized egg.

Optionally, the step of injecting at least one exogenous material isconfigured to keep the at least one delivery mechanism in the designatedblood vessel for 10 seconds.

Optionally, the step of injecting at least one exogenous material isconfigured to apply surgical glue when the at least one deliverymechanism is withdrawn from the designated blood vessel.

Variously, the at least one delivery mechanism comprises at least one ofa group consisting of: syringes, needles, angled needles, jacketedneedles, cantilevered needles, pipettes, multichannel pipettes,tweezers, tubing, magnetic droppers and combinations thereof.

The step of engrafting at least one exogenous material comprises:identifying a junction between a first blood vessel and a second bloodvessel on the chorioallantroic membrane (CAM), and engrafting saidpopulation of cells at the junction.

Variously, the step of configuring the automated sample collectionsystem, comprises defining a configuration parameter selected from agroup consisting of a harvesting date and time; an engrafting date andtime; an injecting date and time; an identified peak time forharvesting; a sample cell extracting days and combinations thereof.

Where required, the method may further comprise analyzing, by at leastone imaging unit, the chorioallantoic membrane (CAM) and the embryo at apre-configured period of time.

Where required, the method may further comprise using, by the at leastone sampler, invasive techniques to collect samples from the at leastone fertilized egg.

Variously, the step of analyzing comprises: using, by the at least oneimaging unit, a non-invasive photographic technique selected from agroup consisting of an imaging fluorescence-based photography technique;an imaging luminescence-based photography technique; an imagingconventional illumination-based photography technique; an imagingultrasonic-based technique; an imaging X-ray based technique; a heatdetector based technique; radioactivity detector based technique;MRI-based technique; and combinations thereof.

Where required, the method may further comprise: using a high-resolutionimaging fluorescence-based photography technique; marking each cell tobe individually identifiable; and indicating an associated cell type byexpressing additional fluorescent labels.

As appropriate, the step of analyzing comprises testing, by the at leastone imaging unit, the convergence of blood vessels towards the graft.

As appropriate, the step of analyzing comprises: testing, by the atleast one imaging unit, for variations in the distribution of density ofchorioallantoic membrane (CAM) blood vessels next to the site of thegraft.

As appropriate, the step of analyzing, comprises: mapping, by the atleast one imaging unit, of blood vessels branching.

Where required, the method may further comprise at least one of:analyzing growth of viruses for generation and testing/development ofvaccines; analyzing of chemotherapies on human or mammalian cancer cellstransplanted to an egg; analyzing for angiogenic properties ofsubstances applied to the chorioallantoic membrane (CAM); analyzing oftoxicity to the embryo (teratogenicity) or transplanted human ormammalian skin transplanted to the CAM including irritation andsensitization.

Where required, the method may further comprise monitoring at least oneactivity selected from the group consisting of: the absorption of thebolus; blood leakage from the blood vessels; bolus ejected from the atleast one fertilized egg; and combinations thereof.

In some embodiments of the current disclosure, an automated biologicalsample collection system is disclosed for collecting biological samplesfrom at least one fertilized egg, the system comprising a set of systemcomponents: at least one incubator configured to control the incubatingenvironment of at least one egg; at least one applicator comprising atleast one delivery mechanism configured to deliver exogenous material toat least one egg; at least one sampler configured to collect at leastone sample from at least one egg; at least one egg supporting apparatusconfigured to accommodate at least one egg; a shell removal apparatusconfigured to remove at least a section of the shell providing access tothe chorioallantroic membrane (CAM); at least one imaging deviceoperable to provide monitoring of system components; and at least onecontroller unit operable to communicate with at least one systemcomponent and drive the system in an automatic manner.

Variously, at least one controller unit is operable to control at leastone factor selected from: incubation conditions, incubation periods,calculation of incubation periods, selection of incubation periods,transfer of eggs from the incubator to the applicator, transfer of eggsfrom the incubator to the sampler, applicator control, detection ofnon-viable eggs, removal of non-viable eggs, image analysis,coordination of feedback from system components, shell removal, sampleractivation, data collection, system configuration and combinationsthereof.

Variously, the system further comprising at least one feedback mechanismselected from a group consisting of: video sensors, light sensors,sonar, shadow contrast detectors, interferometers, piezoelectricelements, a tuning fork and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the embodiments and to show how it may becarried into effect, reference will now be made, purely by way ofexample, to the accompanying drawings.

With specific reference now to the drawings in detail, it is stressedthat the particulars shown are by way of example and for purposes ofillustrative discussion of selected embodiments only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails in more detail than is necessary for a fundamentalunderstanding; the description taken with the drawings making apparentto those skilled in the art how the several selected embodiments may beput into practice. In the accompanying drawings:

FIG. 1A is a schematic diagram representation of an embryonated chickenegg structure;

FIG. 1B is a schematic diagram representation of the blood vesselstructure of the chorioallantoic membrane (CAM) of an embryonatedchicken egg;

FIG. 1C is a schematic diagram representation of the blood vesselstructure including the umbilical vein and the umbilical artery of anembryonated chicken egg;

FIG. 2A is a block diagram showing the main elements of an automatedbiological sample collection system;

FIG. 2B is a block diagram showing the main elements of anotherautomated biological sample collection system;

FIGS. 3A and 3B are schematic cut away isometric projectionsrepresenting an embodiment of a biological sample collection system;

FIG. 4A is a schematic isometric projection representing a furtherembodiment of a biological sample collection system;

FIG. 4B shows a possible shell removal mechanism for use in embodimentsof the biological sample collection system;

FIG. 5A schematically represents a possible applicator for use with thebiological sample collection system;

FIG. 5B schematically represents a possible embodiment of the deliverymechanism being used to inject exogenous material into thechorioallantoic membrane (CAM) of a shell-less egg;

FIG. 6 is a block diagram of a distributed biological sampling systemfor performing assays automatically on embryonated eggs;

FIG. 7 is a block diagram demonstrating possible actions along a timeaxis of incubation days;

FIG. 8 is an illustrative example of different blood vessels branchingonto a chorioallantoic membrane (CAM), observed after implanting;

FIG. 9A is a flowchart representing a method for use with the automaticcollection of biological samples from avian eggs;

FIG. 9B is a flowchart representing another method for use with theautomatic analysis of collected biological samples from avian eggs;

FIG. 9C is a flowchart representing a method of injecting for use withthe automated system for the collection of biological samples;

FIG. 9D is a flowchart representing a method of engrafting for use withthe automated system for the collection of biological samples;

FIG. 9E is a flowchart representing a method of pricking for use withthe automated system for the collection of biological samples; and

FIG. 10 schematically represents a possible embodiment of an egg sealingunit for use with the biological sample collection system.

DETAILED DESCRIPTION

The microcirculation within the chorioallantoic membrane (CAM) of thechick is particularly well suited for in vivo observation and has beenused extensively as an assay to detect angiogenic activity.

Aspects of the present disclosure relate to system and methods of anautomated biological sample collection system using an automated processfor producing biotechnological assays on fertilized and embryonatedeggs.

The embryonated egg is a complex structure and is particularly wellsuited for in vivo observation, utilized as a laboratory host system forvirus research, commercial production of vaccines, detecting angiogenicactivity, special investigations and the like. The embryonated egg iscomprised of a developing embryo and several supporting membranes(chorioallantoic, amniotic, yolk), which enclose cavities or “sacs”within the egg. The developing embryo and its membranes provide thediversity of cell types that are needed for successful replication of awide variety of different viruses, for example. The chorioallantoicmembrane (CAM) is the largest of the embryo membranes enclosing thelargest cavity within the egg, the allantoic cavity. The amnioticmembrane encloses the embryo and forms the amniotic cavity of theembryonated chicken egg. The yolk sac is attached to the embryo andcontains the nutrient that the embryo utilizes during embroynicdevelopment and the immediate post-hatch period.

The automated biological sample collection system comprises an incubatorconfigured to control the incubating environment of at least one egg; anapplicator comprising at least one delivery mechanism configured todeliver exogenous material to a chorioallantroic membrane (CAM) of theat least one egg; a sampler configured to collect samples from the atleast one egg, invasively or non-invasively; at least one imaging deviceoperable to provide monitoring of system components; and at least onecontroller unit operable to communicate with at least one systemcomponent and drive the system automatically.

Description of the Embodiments:

In various embodiments of the disclosure, one or more tasks as describedherein may be performed by a data processor, such as a computingplatform or distributed computing system for executing a plurality ofinstructions. Optionally, the data processor includes or accesses avolatile memory for storing instructions, data or the like. Additionallyor alternatively, the data processor may access a non-volatile storage,for example, a magnetic hard-disk, flash-drive, removable media or thelike, for storing instructions and/or data.

It is particularly noted that the systems and methods of the disclosureherein may not be limited in its application to the details ofconstruction and the arrangement of the components or methods set forthin the description or illustrated in the drawings and examples. Thesystems and methods of the disclosure may be capable of otherembodiments, or of being practiced and carried out in various ways andtechnologies.

Alternative methods and materials similar or equivalent to thosedescribed herein may be used in the practice or testing of embodimentsof the disclosure. Nevertheless, particular methods and materials aredescribed herein for illustrative purposes only. The materials, methods,and examples are not intended to be necessarily limiting.

The Embryonated Egg:

Reference is now made to FIG. 1A, there is provided an embryonated eggstructure diagram, which is generally indicated at 100A, illustratingthe structure of an embryonated egg.

The embryonated egg 100A comprises a developing embryo 52, a shellmembrane 54 (the chorion) is the outermost membrane and lines the insideof the egg outer shell 56; and additional supporting membranes: achorioallantoic membrane (CAM) 58, an amniotic membrane 60, a yolkmembrane 62, which enclose cavities or “sacs” within the embryonatedegg. The chorioallantoic membrane (CAM) 58 is the largest of the embryomembranes enclosing the largest cavity within the egg, the allantoiccavity 64. The amniotic membrane 60 encloses the developing embryo 52and forms the amniotic cavity 66 around the developing embryo 52. Theyolk membrane 62 encloses the yolk sac 68 which is attached to thedeveloping embryo 52.

The embryonated egg 100A further comprises an air pocket 70 which is thespace at the rounded end and has a function in respiration and pressureadjustments. Additionally, the embryonated egg 100A comprises albumin72, which is the egg white and consists mainly of protein.

It is noted that allantoic cavity 64, formed by the chorioallantoicmembrane (CAM) 58, within an egg is a membranous sac which is involvedin gas exchange, storage of wastes and absorption of nutrients for thedeveloping chick embryo 52; the amniotic cavity 66 is filled with liquidand serves to protect the developing embryo 52 against physical damageand is also functioning as an area of exchange of molecules. Theamniotic membrane 60 is the innermost membrane which encloses thedeveloping embryo to provide a protective environment to the developingembryo 52 of the embryonated chicken egg.

Reference is now made to FIG. 1B, there is provided a blood vesselstructure diagram, which is generally indicated at 100B, illustrating ablood vessel structure of the chorioallantoic membrane (CAM) of anembryonated chicken egg.

The blood vessel structure diagram 100B illustrates the formation of asupportive matrix for the extensive vascular network that crossesthrough the chorioallantoic membrane (CAM) growing during incubation.The diagram 100B is illustrating a branching network of large and smallblood vessels over the major cavities (“sacs”) within the embryonatedegg: the allantoic cavity 64 includes the allantoic blood vesselsbranching 75, the amniotic cavity 66 around the developing embroy 52 andthe yolk sac 68 which includes the vitelline blood vessel branching 76.As appropriate, the vitelline arteries are the arterial counterpart tothe vitelline veins. Like the veins, they play an important role in thevitelline circulation of blood to and from the yolk sac 68.

It is noted that the chorioallantoic membrane (CAM) develops in a shorttime from a small vascular membrane into a structure that covers theentire inner surface of the shell displaying a densely organizedvascular network. The blood vessel network includes veins which areresponsible for transporting blood throughout the body, arteries andcapillaries that carry blood away from the body and exchange nutrients,waste, and oxygen. Further, closest to the developing embryo 52 is theprimary stratum which is the thickest part of the chorioallantoicmembrane (CAM), approximately 20 to 100 micrometer thick, and thelargest chorioallantoic membrane (CAM) vessels (up to approximately 1 mmin diameter) run immediately underneath and are anchored to this layervia thin, perivascular sheathes. Smaller vessels (less than 200micrometer diameter) are embedded within the main layer. Immediatelyexternal to the primary layer of chorioallantoic membrane (CAM) is acapillary plexus, or blood sinus. Above this sinus lies a thin stratum(approximately 1 micrometer) that is composed of four sublayersoriginally derived from the chorion (basement membrane, epithelial celllayer, peptidoglycan extracellular matrix, and basal lamina).

Accordingly, the identification of the various blood vessel (veins,arteries, capillaries) may be useful for the process of injectingexogeneous material, as part of an assay, as elaborated in sectionshereinafter.

Reference is now made to FIG. 1C, there is provided another view of theblood vessel structure diagram, which is generally indicated at 100B,illustrating additionally the umbilical vein and the umbilical artery ofan embryonated chicken egg.

The view 100C of the blood vessel structure of an embryonated chickenegg includes the egg shell 56 protecting the embryo 52 and illustratesspecifically the umbilical vein 82 and the umbilical artery 84 of theembryonated egg.

The Automated System:

The automated biological sample collection system is operable to conductbiological assays, using embryonated eggs, according to a pre-configuredspecification and carry out the assay schedule automatically. Theautomation of the biological sample collection system includes automaticmechanical operation controllable via a system controller and furtherconfigured to analyze the collected samples automatically using varioussystem components and software techniques. The system is operable toperform clinical assays as well as providing answers to the pharmaindustry.

Reference is now made to FIG. 2A, there is provided an exemplified blockdiagram of an automated biological sample collection system, which isgenerally indicated at 200A, for performing assays on fertilized orembryonated eggs. The automatic sample collection system block diagram200A represents the main elements of one system embodiment and includesan incubator 120 for incubating an egg, an applicator 140 for applyingexogenous material to the egg, a sampler 160 for collecting biologicalsamples from the egg (an egg sealing unit, see FIG. 10) an egg supportapparatus 180 and a controller 110 operable to coordinate the system.

It is a feature of the embodiment of the biological sample collectionsystem 200A that it may be used to automate biological screening,particularly of fertilized avian eggs. Consequently, the system 200A mayincrease the throughput of biotechnological sampling thereby enablinglarge scale performance of biotechnological assays.

A biological sample collection system 200A of the type disclosed hereinmay be usefully applied to a variety of technologies. For example, therapid sampling may make it practical to grow viruses in avian eggs whichare harvested to generate vaccines.

In other applications, human or mammalian cancer cells may betransplanted to eggs. These chimeric systems may be used to performassays of treatments such as chemotherapies, radiotherapies and thelike. This technique may be used for drug development applications, forpersonalized medicine or the like.

Assays may be performed upon various microbes, cells, tissues, organsections or the like. These may be injected into, grafted upon orotherwise applied to the embryo or fertilized egg. It is particularlynoted that assays may be performed upon the chick embryos or upon thechorioallantoic membrane (CAM) of the eggs.

Alternatively, or additionally, assays may be performed for angiogenicproperties of substances applied to the chorioallantoic membrane (CAM)or for toxicity to the embryo (teratogenicity) or human or mammaliantissues transplanted to the chorioallantoic membrane (CAM). Such assaysmay be used to examine irritation, sensitization and the like.

Still further applications will occur to the skilled practitioner.

Reference is now made to FIG. 2B, there is provided another exemplifiedblock diagram of an automatic biological sample collection system, whichis generally indicated at 200B, for performing assays on fertilized orembryonated eggs. The automatic sample collection system block diagram200B represents the main elements of another system embodiment andincludes an incubator 120 for incubating an egg, an applicator 140 forapplying exogenous material to the egg, a sampler 160 for collectingbiological samples from the egg, an egg support apparatus 180 driven bya driver unit 182 and a controller 110 operable to coordinate thesystem. As appropriate, the sampler 160 includes an imaging unit 162, asample extractor 164 and a sample analyzer 166, as detailed in the “TheSAMPLER” section.

It is noted that the imaging unit 162 associated with the sampler 160 ispresented by example only. Additional imaging devices may be associatedwith other system elements such as with the applicator 140, egg sealingunit (see FIG. 10) and the incubator 120 to provide better automatedtools for performing the assays' analysis.

The Incubator:

The incubator 120 of the automatic biological sample collector system200A (FIG. 2A) is configured to control the environmental conditions ofthe eggs. Typically, the incubator 120 maintains environmentalconditions selected to support the viability of the fertilized eggs.Furthermore, the incubator may be adapted to control variousenvironmental parameters such as ambient temperature, humidity, aircirculation, anti-microbial agent content, ambient pH, egg-turning,pressure, gaseous composition, oxygen content and the like. In eggswhere the blood vessels are poorly developed on the CAM, for examplefollowing prolonged incubation at low temperatures that halt embryonicdevelopment (e.g., 14-20 Celsius), a decrease or increase in air oxygenlevels at the CAM surface may induce more robust blood vesseldevelopment. Oxygenated air (for example 15-35% oxygen) may beintroduced directly to the CAM via the prick hole in the egg using anaspirating needle, for example, or by altering oxygen levels in theentire incubator. The incubator may include a convection heater 122, awater reservoir, for maintaining humidity, an air circulation system 124such as a fan, as well as regulators 126 operable to maintain conditionswithin selected ranges. It is further noted that the egg supportapparatus 180 may be adapted to turn the eggs during incubation, forexample, by tilting a tray repeatedly to simulate the motion of an eggincubated in a natural environment.

The automated biological sample collection system 200A (FIG. 2A) may beadapted to a variety of types of eggs. Typically, chicken, turkey,ostrich, quail, duck, pheasant, grouse, or other bird eggs may be used.It is noted that while the incubation period for chick eggs is 21 days,turkey eggs have longer incubation periods of 28 days while ducks mayhave incubation periods of up to 42 days depending on the species. Thusthe species of the host eggs may be selected to suit requirements, forexample such that implanted cells are maintainable for longer.

In various embodiments, the automated biological sample collectionsystem 200A (FIG. 2A) may be used to collect samples from modified avianeggs wherein at least a portion of the egg shell has been removed fromthe fertilized avian egg. In these embodiments, the egg supportapparatus 180 typically includes receptacles adapted for holding atleast partially shell-less fertilized eggs. A number of referencesdescribe well-known protocols for incubating shell-less, fertilizedavian eggs (Hamamichi and Nishigori 2001. Toxicol. Lett. 119: 95-102;Fisher, C. J. 1993. Chick Embryos in Shell-less Culture, Pages 105-115,in Tested studies for laboratory teaching, Volume 5 (C. A. Goldman, P.L. Hauta, M. A. O'Donnell, S. E. Andrews, and R. van der Heiden,Editors). Proceedings of the 5th Workshop/Conference of the Associationfor Biology Laboratory Education (ABLE); Tufan et al 2004. Akdogan EsatAdiguzel Neuroanatomy, 2004, 3 8-11). Particular reference is made toUnited States patent application publication number US 2009/0064349,which is incorporated herein by reference. US 2009/0064349 disclosesvarious methods of cultivating transplanted skin explants onto thechorioallantoic membrane (CAM) of fertilized avian eggs. None of thesereferences disclose, or suggest, an automated process for samplecollection.

The ambient temperature of incubation is typically within the range of30 to 40 degrees Celsius such that viable embryos may be maintained. Theselected temperature may depend upon other requirements, for example,without wishing to be limited to any particular theory, it isparticularly noted that for various applications a temperature of 40degrees Celsius may be used to increase the effectiveness of chemicalinhibitors of tumor growth while supporting the viability of the hostembryo (Taylor and Williams, Biochemistry vol. 42, 1956 pp. 57-60).

According to various embodiments, a plurality of incubators may beprovided, or a plurality of compartments within an incubator, such thatvarious eggs may be subjected to different environmental conditionsduring incubation. Additionally or alternatively, the incubator may beadapted to provide gradual variation of environmental conditions, theeffects of which may be tested.

In some embodiments, a vitality monitor 128 may be provided which isoperable to monitor the eggs during incubation for signs of life fromthe egg. Such signs of life may include an embryonic heartbeat, pulse,blood flow, staining with and detection of vitality dyes or the like.According to one embodiment, the implanted cells may be labeled tomeasure proliferation by dilution, in which the count per cell decreasesalthough the overall count remains the same. For example, a cyanine dye,such as DiI may be used for fluorescently labeling membranes, ortritiated nucleotides may be used for radioactively labeling DNA. Otherlabels may permanently label cells, such as viral infection with alentivirus expressing a fluorescent tag such as GFP, or an enzymatic tagsuch as luciferase. Such genetically encoded labels can be regulated bygene promoters or protein degradation tags such that their expressionreflects intracellular metabolic events, such as cell cycle activity,mitochondrial membrane potential, protein synthesis etc. These labelscan be measured by a vitality monitor 128 adapted to perform, amongother techniques, fluorescence microscopy for DiI, GFP or the like, orMRI for tritiated probes or the like, and a luminescence counter forluciferase when the luciferin substrate is added to the exogenousmaterial in the egg.

It will be appreciated that the vitality monitor may allow non-viableeggs to be identified early. Optionally, an egg removal system may beprovided to remove such eggs from the biological collection system 200A(FIG. 2A).

It is noted that the period of incubation may be selected to besufficient to allow engraftment of the population of cells to thechorioallantoic membrane (CAM) of the at least one fertilized egg.

The Applicator

The applicator 140 is configured to deliver exogenous material to theeggs. The applicator typically includes a delivery mechanism 142, amanipulator 144 and a feedback mechanism 146.

The delivery mechanism 142 is configured to deliver the exogenousmaterial to the eggs. Exogenous material to be applied may be an implantsuch as a cell culture administered to the egg. For example, implantsmay include tumor cells, skin explants, biopsied material or the like.Such implants may be applied to fertilized eggs following an initialincubation period. Alternatively, or additionally, other exogenousmaterial may include foreign bodies such as bacteria, viruses orexternal modulators.

As suits requirements, the delivery mechanism 142 may be adapted toinject the exogenous material into a compartment of the egg such as theamnion or yolk sac, or into a blood vessel of the egg, the embryo or thechorioallantoic membrane (CAM). Additionally or alternatively, thedelivery mechanism 142 may be adapted to apply the exogenous materialdirectly onto the chorioallantoic membrane (CAM).

It is noted that, as outlined below, in order to improve engraftment inthe egg, the egg immune response may be reduced by irradiating, usingX-rays, gamma rays or the like, prior to engraftment of the implantedcells. Accordingly, X-ray or gamma ray sources may be incorporated intothe system, perhaps in compartments isolated by lead lining or the like.Alternatively, the system may be adapted such that where required, eggsmay be removed and irradiated, perhaps automatically, as necessary. Thesystem may be further configured to administer immunosuppressant drugs.

Accordingly, various applicators 140 may include delivery mechanisms 142such as syringes, needles, cantilevered needle, jacketed needles,pipettes, multichannel pipettes, tweezers, magnetic droppers or thelike.

Optionally, a reservoir 148 may be provided for storing the exogenousmaterial before application to the egg. A reservoir may be providedincluding an agitator such as a stirrer or the like in order to maintaina constant composition of an agent to be delivered to the egg.Alternatively, or additionally, a reservoir may include an array ofwells for containing agents of predetermined composition. For example,an 8×12 array of wells may contain material to be delivered by an eightchannel pipette into an array of eggs. It will be appreciated that otherconfigurations may occur to the practitioner. The material to be storedin the reservoir may be in liquid or in solid form. The reservoir may beused to form gel “plugs” (see “Identification and Delivering” sectionbelow) containing exogenous material or external modulators.

A label may be added to the exogenous material or external modulators,for example to assess whether the matter has entered the correctcompartment of the egg upon delivery by the applicator. Thus forintravenous injection, a dye such as fast green may be added, or a bloodvessel staining dye such as fluorescent dextrans, or a blood vesselrestricted dye such as Evans Blue.

According to various embodiments, external modulators may include, butare not limited to, chemical agents, biological agents, radiation,mechanical aggression, thermal stress, contact sensitizers, allergens,gaseous agents, mechanical barriers and the like. Biological agents mayinclude living cells or organisms, such as immune cells (including butnot limited to T-cells, B-cells, NK cells and myeloid cells) ormicrobes.

Chemical agents may include surfactants, retinoids, carotenoids, foodadditives, moisturizers, mustard gas, organic molecules, inorganiccompounds, vitamins, UV absorbing agents, UV protecting agents,perfumes, cosmetic formulations, lacquers, glues, paints, colorants,detergents, balms, creams, dyes, hair dyes, emulsions, gels, greases,shake lotions, pastes, oils, liposome formulations, lotions, mousses,ointments, suspensions, aqueous solutions, salves, solvents, shampoos,pollutants, steroids, shower gels, antibiotics, sulfa drugs,antiseptics, disinfectants, herbal formulations, anti-inflammatoryagents, pharmaceutical compositions, aerosols, cleaning products,powders, petroleum jelly, anti-microbial agents, soaps, nail polish,acid rain, gasoline, kerosene, alcohols, industrial solvents, causticchemicals, herbicides, metals, chelating agents, pesticides, mediations,fumigants, insecticides, fungicides, cleaning materials, contactsensitizers, allergens, impregnated dressings, solutions, occulativedressings, compression dressings, gallium compounds, kinase modulatingagents, phosphate buffered saline (PBS), enzyme inhibitors, protease,secretions of plants and animals, endotoxins, antimicrobialformulations, tazarotene formulations, bexarotene formulations, azoleformulations, topical antibiotic formulations (e.g. tetracyclinefamily), plant derived toxins (e.g. poison ivy), animal-derived toxins,putative ameliorative agents (e.g. aloe vera), depilatory or hairgrowth-enhancing reagents, putative anti-aging formulations and the likeas well as combinations thereof.

Biological agents may include, but are not limited to, hormones (e.g.estrogens), peptides, cytokines, chemokines, interferon formulations,nucleic acids, proteins, carbohydrates, carotenoids, lipids, fattyacids, prions, enzymes, lectins, antibodies and the like as well ascombinations thereof.

In some embodiments the exogenous material may consist of animmobilising agent that is injected around the vicinity of the bloodvessel that has been selected for injection by the applicator. Theimmobilising agent reduces the natural movement of the blood vessel, aswell as providing contra to the impact of the injecting needle orcatheter. The immobilising agent may comprise a cross-linking chemicalthat hardens albumen, for example.

Following application of exogenous material, the eggs together with theexogenous material may be returned to the incubator for an additionalperiod for continued incubation. During this second period, anastomosisof the host and implant blood vessels may occur, where there are extantblood vessels in the engrafted implant material. For exogenous materiallacking extant blood vessels, the chick vasculature generates a de novoblood vessel network in the impalnted material. The implant is typicallynourished by the chick's blood and gases exchanged by chickerythrocytes. It may therefore be necessary to isolate the implant frommicrobes in the ambient atmosphere outside of the egg shell which couldcontaminate the incubating implant and possibly lead to tissuedegradation. Possible means for isolating the implant or sealing the egginclude, but are not limited to, placing of an egg sealing unit (seeFIG. 10), covering with saran wrap, adhesive tape, the egg shell itself,glass-coverslips sealed with wax, providing a sterile environment andthe like.

In some embodiments, the environment provided during the secondincubation period may differ from that during the initial incubationperiod, for example, by selecting chemical compositions or exposure toradiation which may influence the development of the implants such thatenvironmental conditions may be tested.

The manipulator 144 is provided to manipulate the delivery mechanism 142relative to the egg. This is necessary in order for the deliverymechanism 142 to bring the exogenous material to the desired region ofthe egg without damaging the egg. For example, where the deliverymechanism 142 is required to inject material into a blood vessel, themanipulator 144 is operable to direct the injection head of the deliverymechanism to the relevant blood vessel and at the required angle ofattack. Similarly, where a cell culture is to be applied to thechorioallantoic membrane (CAM), for example, the manipulator 144 isoperable to bring the delivery mechanism 142 into the desired regionwithout damaging the surrounding tissue.

Various manipulators may be utilized in embodiments of the automatedbiological sampling system 200A (FIG. 2A). These includemicromanipulators, robotic arms, articulated arms, telescopic arms,tracks, runners and the like as well as combinations thereof.

According to various embodiments, the manipulator 144 may be operable toact upon the delivery mechanism 142, moving it into position relative tothe egg. Alternatively, the manipulator 144 may be operable to move theegg into position relative to the delivery mechanism 142, possibly bymanipulating the egg support apparatus 180.

In order to control the movement of the manipulator 144, the feedbackmechanism 146 is operable to provide feedback. The feedback mechanism146 typically includes at least one sensor configured to sense theposition of the delivery mechanism 142 relative to the egg, and aprocessor operable to direct the manipulator 144 to the desiredposition. Various feedback sensors may be used for such an arrangement;these include video sensors, sonar, shadow contrast detectors,interferometers, piezoelectric elements, tuning forks and combinationsthereof.

For example, a CCD camera may be linked to a feedback system to controla robotic arm manipulating the delivery system. Fine control in theapproach to the target region may be supplemented by interferometricanalysis of a laser beam reflected from the surface of the targetregion, alternatively or additionally, a piezoelectric element attachedto at least one tine of a tuning fork may be used to monitor changes inthe resonant frequency of the tuning fork as it approaches the surface.Other feedback mechanisms may be preferred as suit requirements.

The Shell Removal Mechanism:

In some embodiments, the system 200A (FIG. 2A) may further include ashell removal mechanism. The shell removal mechanism is provided toremove at least a section of the shell in order to provide access to thechorioallantoic membrane (CAM) and the embryo within.

Where only a section of the shell is to be removed, the shell removalmechanism may include a cutter for slicing a ring around the egg, and asucker for removing the cut ring. Where a shell-less egg is required theshell removal mechanism may be configured to break the fertilized egginto a suitable egg support apparatus 180.

Optionally, the shell removal mechanism comprises an electric drill toexpose the chorioallantoic membrane (CAM) delineated vascular system.

It is noted that only eggs on which a distinct fine vascular system canbe recognized on the chorioallantoic membrane (CAM) is suitable fortesting. This is sometimes considered a critical criterion to develop asuccessful assay, and may lead to a selection procedure, where required.

To improve imaging or visualization of the CAM blood vessels, a smallarea of CAM itself may be removed, since it is opaque and interfereswith light transmission, reducing accuracy of injections and imaging andthe like. To prevent leakage of egg material upon CAM section removal, asealing gel may be applied to the hole in the CAM, whose properties arecompatible with needle penetration and resealing after removal, as wellas being mostly translucent for accurate imaging and needle guidance.

The Egg Support Apparatus:

The egg support apparatus 180 may be provided in order to hold the eggsin the sample collection system 200A (FIG. 2A). Accordingly, the eggsupport apparatus 180 is typically a tray having an array of stands forsupporting the eggs within the system 200A (FIG. 2A). As noted above,where shell-less eggs are to be incubated, the egg support apparatus 180may have suitably adapted receptacles, for example cups with covers.Optionally, such receptacles may be disposable.

According to some embodiments, the egg support apparatus may beconstructed from transparent material, such as glass, PVC or the like.It will be appreciated that such a construction would allow imaging ofthe eggs contained by the egg support apparatus from various angles.

Apart from containing the eggs within the system 200A (FIG. 2A), the eggsupport apparatus 180 may have additional functionality as required. Forexample, the egg support apparatus may be adapted to provide a turningmotion to the eggs during incubation.

Typically, the egg support apparatus 180 is adapted to move betweenvarious zones of the system 200A (FIG. 2A). For example, a traysupporting an array of eggs, may be mounted upon a rail and adapted tomove between separate compartments or chambers for incubation,application of exogenous material and/or sampling.

Furthermore, the egg support apparatus 180 may itself form part of themanipulator 144 for positioning the egg relative to the deliverymechanism 142.

Optionally, the egg support apparatus 180 is controllable via a driver182 (FIG. 2B, FIG. 6) operable to receive commands via the userinterface (FIG. 6, item 118) of the system controller (FIG. 2A, FIG. 2B,FIG. 6 item 118).

The Sampler:

The sampler 160 is provided to collect biological samples from the eggs.Sampling may be invasive, possibly even destructive to the egg.Additionally or alternatively, non-invasive or remote sampling may bepreferred.

For example, material may be removed invasively for analysis, biopsy,assay and the like. Removed material may be used in biological analysistechniques such as, but not limited to, Polymerase Chain Reaction (PCR),Fluorescence-Activated Cell Sorting (FACS), Flow Cytometry (FCM),Enzyme-Linked Immunosorbent Assay (ELISA), Enzyme-Linked ImmunosorbentSpot (ELISPOT), Enzyme Multiplied Immunoassay Technique, RAST test,Radioimmunoassay, Immunohistochemistry, Immunofluorescence, dyeabsorption, enzymatic activity and the like. Chemical analyses of thesampled material may include spectrophotometry, HPLC, mass spectrometry,for determining the concentration of external modulators in the sampledmaterial. Indeed, where required, the system 200A (FIG. 2A) mayincorporate any or all of these material analysis devices.

Alternatively, samples may be obtained non-invasively using imagingtechniques such as photographic imaging, radiographic detection,Magnetic Resonance Imaging (MRI), fluorescence imaging, luminescenceimaging, ultrasonic imaging, heat detection, x-ray imaging,scintigraphy, Positron Emission Tomography (PET), photoacoustic imaging,thermography, and the like as well as combinations thereof. Indeed,where required, the system 200A (FIG. 2A) may incorporate any or all ofthese non-invasive sampling devices.

It will be appreciated that non-invasive sampling may be useful,particularly where ongoing monitoring is required. For example, thesampler 160 may be configured to periodically take measurements from theegg during incubation.

Still another method of sampling may be to install a catheter or similardevice into a blood vessel or one of the liquid compartments with avalve that can be opened, in order to take multiple samples. Forexample, a needle attached to fine silicon tubing may be inserted into achorioallantoic membrane (CAM) vessel, with a valve that can be openedfor sampling blood at desired intervals.

Detailed Projections:

In order to further illustrate the various embodiments of the system,reference is now made to FIGS. 3A and 3B which show schematic cut awayisometric projections representing one embodiment of a biological samplecollection system 1100.

The embodiment includes an incubation chamber 1120, an applicationchamber 1140 and a sampling chamber 1160. The egg support mechanism 1180includes multiple trays 1182 a-d mounted upon tracks 1184 a-d.

The egg support trays 1182 a-d are provided to contain fertilized eggs.In the example provided in FIGS. 3A and 3B, each tray includes a 9×8array of egg receptacles 1186. Where appropriate, the receptacles may beconfigured to contain complete eggs, shell-less eggs or partiallyshell-less eggs. It will be appreciated that the 9×8 array of theexample may be of particular use where an eight channel syringe is usedto apply exogenous material to the eggs, for example, when such matteris applied manually. Other configurations may be preferred as suitrequirements.

The trays 1182 a-d are mounted on tracks 1184 a-d which extend throughall three chambers 1120, 1140, 1160 and along which the trays may bemoved. With particular reference now to FIG. 3A, showing the automatedsystem 1100 during one of the incubation phases, all the trays 1182 a-dare arranged in the incubation chamber 1120.

The incubator is configured to keep the incubated eggs at a temperatureof approximately between 30 to 40 degrees Celsius so as to maintain anenvironment suitable for the continued viability of fertilized eggs. Itwill be appreciated that a turning mechanism (not shown) may be providedto periodically tilt the trays to simulate the turning of eggs duringnatural incubation.

Reference is now made to FIG. 3B, there is provided the automatedsystem, which is generally indicated at 1100, showing an alternativeconfiguration; the one tray 1182 d has been transferred, along itsassociated track 1184 d, to the application chamber 1140, forintroduction of exogenous material to the eggs and another tray 1182 chas been transferred to the sampling chamber 1160.

In the application chamber 1140, a delivery mechanism 1142 mounted to amanipulator 1144 may be directed to a selected egg within the tray 1182d. The manipulator 1144 of the example includes a roof mountedtelescopic arm configured to move the delivery mechanism 1142 to thedesired position within the application chamber. It will be appreciatedthat other manipulators may be additionally or alternatively provided asrequired.

The tray 1182 c is shown transferred to the sampling chamber 1160wherein it may be inspected using an imager 1162. Various imagers 1162may be utilized in the automated system 1100 as outlined herein.Additionally or alternatively, material may be collected from the eggsfor analysis.

In order to prevent movement of the embryo during application of theexogenous material, the application chamber 1140 may be cooled to a lowtemperature such as around 18 degrees Celsius or so. Accordingly, aninsulating partition (not shown) may be provided between the incubationchamber 1120 and the application chamber 1140.

The application chamber 1140 may be adapted for other methods forpreventing movement such as applying gentle suction to the membrane ordropping sterile filter paper rings onto the chorioallantoic membrane(CAM) to maintain stretched blood vessels for injection/removal ofblood.

Following application of the exogenous material, the tray 1182 d may bereturned to the incubation chamber for additional periods of incubation.The process may be repeated a plurality of times with differentexogenous materials being applied. For example, an incubated egg may betransferred to the application chamber 1140 initially for application ofan implant or the like. Then, the egg may be returned to the incubationchamber 1120 for further incubation until the engraftment of the implantoccurs. The egg may be returned to the application chamber 1140,following re-incubation so that an external modulator, such as describedabove, may be applied to the egg. The egg may be transferred again tothe incubation chamber 1120 for further incubation during which the eggmay be periodically transferred to the sampling chamber 1160 forexamination.

It will be appreciated that the three chambered system 1100 describedhereinabove in relation to the embodiment of FIGS. 3A and 3B representsonly an example of a possible biological sampling system. Alternatively,other embodiments may be preferred to suit different requirements.

Reference is now made to FIG. 4A, there is provided a schematicisometric projection representing a further embodiment of an automatedbiological sample collection system, which is generally indicated at2100. The automated system 2100 of the embodiment of FIG. 4A includes anincubator compartment 2120, a second compartment 2150 and an egg supportapparatus 2180.

The incubator compartment 2120 has a convection heater 2122 and an aircirculator or filtration system or the like. The second compartment 2150is a multifunctional space which may include various mechanisms such asmanipulators 2144 a, 2144 b, samplers 2162 a 2162 b, shell removalmechanisms (not shown) and the like. Partitions 2170 may be provided todivide the separate compartments; such partitions may be insulatedand/or sealed to allow the separate compartments 2120, 2150 to supportdifferent environmental conditions.

The egg support apparatus 2180 of the embodiment of FIG. 4A includesmultiple egg trays 2182 a-c coupled to a central support column 2184 ina manner that allows rotation. The trays 2182 a-c are configured toindividually rotate around the central support column 2184 such thateach tray may be transferred from the incubator compartment 2120 to thesecond compartment 2150. Optionally, the trays 2182 a-c may be furtherconfigured to rotate about their central axes to position the eggs 20supported thereby.

In the example, a first tray 2182 a is shown supporting complete eggs 20during an incubation phase. Following an initial incubation period, thetray may be transferred to the second chamber 2150 where a shell removalmechanism, such as described herein below in relation to FIG. 4B, may beused to remove a section of the egg shell, and the applicator may beused to apply exogenous material to the egg. A second tray 2182 b isshown supporting eggs 22 which have had the top section of their shellsremoved, typically, such eggs may be returned to the incubator for asecond incubation period following the application of exogenousmaterial.

The third tray 2182 c is shown supporting eggs 24 following a secondincubation period after which the eggs 24 have been transferred into thesecond chamber 2150 for application of additional exogenous material,for example, an external modulator such as described hereinabove or thelike.

Reference is now made to FIG. 4B, there is provided a possible shellremoval mechanism, which is generally indicated at 30, for use withembodiments of the automated biological sample collection system. Theshell removal mechanism 30 includes two manipulators 32, 34. The firstmanipulator 32 has a suction head 36 operable to engage the top section26 of the egg shell of an egg 20. The second manipulator has a bladehead 38 configured to sever the top section 26 from the egg 20.Optionally, each egg may be rotatable individually to further manipulatethe egg's position. Other shell removal mechanisms 30 are known and mayoccur to the skilled practitioner.

Reference is now made to FIG. 5A, there is provided a schematicrepresentation of a possible applicator, which is generally indicated at3140, for use with an automated biological sample collection system anda tray 3182 of receptacles for containing shell-less eggs. Theapplicator 3140 includes a delivery mechanism 3142, a manipulator 3144,an illuminator 3143, a detector 3145 and a feedback mechanism 3146.

The tray 3182 contains egg receptacles 3186. The egg receptacles 3186are preferably made of a material transparent to the type of radiationemitted by the illuminator 3143 such that the detector 3145 is able tomonitor the relative positions of the delivery mechanism 3142, theorgans of the shell-less eggs and their embryos contained within thereceptacles 3186.

The detector 3145 is typically in communication with the feedbackmechanism 3146 which is operable to direct the delivery head 3142accordingly.

Reference is now made to FIG. 5B, there is provided a possibleembodiment of the delivery mechanism, which is generally indicated at3142, for delivering exogenous material. The delivery mechanism 3142includes an angled needle 3145 which may be used to inject the exogenousmaterial into the chorioallantoic membrane (CAM) of a shell-less egg 26or other organs or blood vessels.

The Controller:

Referring back to the block diagram of FIG. 2A, in order to control andcoordinate the automated biological sample collection system 200A, acontroller 110 may be provided. The controller 110, which may be incommunication with the various functional elements, may be operable toperform a sequence of steps according to an algorithm and support therequired automation.

Accordingly, the controller 110 may be functional to coordinate stepssuch as incubation conditions, incubation periods, calculation ofincubation periods, selection of incubation periods, transfer of eggsfrom the incubator to the applicator and/or sampler, applicator control,detection of non-viable eggs, removal of non-viable eggs, imageanalysis, coordination of feedback for the manipulator, shell removal,sampler activation, data collection and the like.

Where appropriate the controller 110 may include a processor, adatabase, a memory and/or a user interface as required. The controller110 may further be operable to support remote communications asillustrated in FIG. 6, hereinafter.

Reference is now made to FIG. 6, there is provided a block diagram of adistributed biological sampling system, which is generally indicated at600, for performing assays automatically on embryonated eggs. Thedistributed biological sampling system 600 includes a system controller110 operable to automatically control and monitor the system, where thesystem controller 110 is in communication with the incubator 120, theapplicator 140 and the sampler 160. The system controller 110 comprisesa processor 112, a database 114, and a memory 116 and operable tocommunicate with the various external elements via a user interface 118.

The system controller 110 is further operable to communicate with thedriver 182 which is configured to drive the egg support apparatus 180according to assay specifications.

Furthermore, the system controller 110 may be operable to communicateremotely with a server 150 via a communication network 130 such as theInternet. This may support controlling and monitoring the automatedsampling system centrally and further perform the required analysis ofthe sample or associated imaging.

Where appropriate, the controller software is operable to configuresystem settings such as, harvesting date and time; an engrafting dateand time; an injecting date and time, and the like.

The controller software is further operable to identify the peak timefor harvesting and the experimental mode may be used to determine daysupon which the sample cell may be extracted.

As appropriate, the system controller is operable to check if the drugapplied is absorbed, which may be executed in various ways, such asmonitoring the absorption of the bolus by monitoring blood leakage froma blood vessel or detecting a bolus ejected from the egg, for exampleusing dyes and the like that have been added to the exogenous materialor external modulators, prior to injection or delivery. Additionally oralternatively, the delivered drug may be labeled such that its arrivalat the target cells may be confirmed. Other secondary effects may bemonitored as will occur to those in the art. For example, cell membranemovement is affected by chemotherapy agents within minutes of drugapplication, and can be monitored using fluorescence imaging offluorescently labeled cell membranes.

In such cases where a failed drug delivery is observed, it may bedesirable to reject the egg or to retry the injection as appropriate.

Blood Vessel Detection:

The chick embryo has been extensively used to answer very differentquestions at different stages of development. The avian embryo developsa structure for gas exchange with its environment that has some of thesame functions as the mammalian placenta. The chorioallantoic membrane(CAM), with its capillary bed, is the respiratory organ of the chickembryo until the 19th day of incubation, at which time the embryo pipsinternally in the air cell and starts air breathing, thus it has becomean attractive model for study and research.

The circulation in the chick embryo is comparable to that of themammalian fetus. The left and right chorioallantoic arteries bring thedeoxygenated blood into contact with the chorioallantoic membrane (CAM)where gas exchange through the egg shell occurs and the chorioallantoicvein returns the oxygenated blood to the embryo. These vessels areequivalent to the umbilical circulation. After opening the air cell andthe extra embryonic membranes, by a shell removal mechanism, thesevessels are easily accessed. Further, using various automatedtechnologies using transducers and imaging the veins appropriate forinjection may be exposed.

Optionally, the shell removal mechanism comprises an electric drill toexpose the chorioallantoic membrane (CAM) delineated vascular system.

It is noted that only eggs on which a distinct fine vascular system canbe recognized on the chorioallantoic membrane (CAM) is suitable fortesting. This is considered a critical criterion to develop a successfulassay.

It is further noted that the sampler when placed where thechorioallantoic membrane (CAM) is to be assessed should be illuminatedin such a way to avoid shadows and high temperatures for the eggs.

The blood vessels of the embryo may be identified, in particular themain vessel, the umbilical vein which is connecting the chorioallantoicmembrane (CAM) and the embryo. It is known that blood flows fromchorioallantoic membrane (CAM) to the embryo through the umbilical vein.This may facilitate detection of the direction of blood flow. If theumbilical vein is not always connected to the air pocket (FIG. 1A, item70) then it may be useful to move the air pocket to the position of theumbilical vein.

Additionally, shining a light through the shell may allow to see thepositions of other blood vessels. Identification of other blood vesselscarrying blood towards the chorioallantoic membrane (CAM) may be usefulas it allows exogenous material to be injected in the direction of thechorioallantoic membrane (CAM) directly without the material firstpassing through the embryo. This is particularly important wheninjecting “T-Cells” into veins as such T-cells may be large and prone togetting stuck in the capillaries of the embryo, thus it is important todetermine whether blood flow is to the chick body or to the CAM, as wellas determining the loss of exogenous T-cells in the blood due to theirinability to pass through chick capillaries or organs (e.g., liver), ineach direction.

Injection into arteries generally causes bleeding that can lead to lossof chick viability and leakage of the external modulators or exogenousmaterial being injected. Injecting into veins causes much less damage tothe chick or loss of injected material. By injecting in the direction ofthe blood flow there is less resistance to the injection pressureapplied by the applicator, and thus less chance of damage to theinjected blood vessel. Slow, continuous injection of material furtherimproves blood vessel recovery following injection. In one embodiment,an intravenously inserted catheter connected to a pump to injectmaterial in the direction of venus blood flow, may provide theappropriate conditions for achieving minimal loss in chick viability andmaximal delivery of injected material.

The diagrams of FIG. 1B, hereinabove and FIG. 8 hereinafter,illustrating a blood vessel network and other organs of importance forperforming engraftment and injections of the exogenous material into thechorioallantoic membrane (CAM) or into other organs of an embryonatedegg.

Analysis:

The software of the system controller is operable to perform the variousaspects of a desired assay, using automatically controlled componentssuch as an imaging unit (FIG. 2B), feedback sensors, mechanical elementsand the like.

Various feedback sensors may be used for such an arrangement; theseinclude video sensors, sonar, shadow contrast detectors,interferometers, piezoelectric elements, tuning forks and combinationsthereof.

For example, a CCD camera may be linked to a feedback system to controla robotic arm manipulating the delivery system. Fine control in theapproach to the target region may be supplemented by interferometricanalysis of a laser beam reflected from the surface of the targetregion, alternatively or additionally, a piezoelectric element attachedto at least one tine of a tuning fork may be used to monitor changes inthe resonant frequency of the tuning fork as it approaches the surface.Other feedback mechanisms may be preferred as suit requirements.

Reference is now made to FIG. 7, there is provided the block diagram,which is generally indicated at 700, demonstrating possible actionsalong a time axis of incubation days.

The block diagram 700 illustrates an initial incubation period A of aduration according to the requirements of a specific assay. The initialincubation period may be followed by a pricking step 702, which may befollowed immediately by a delivering step 704. Alternatively, thedelivering step may be delayed for a time duration, according to theassay specification and requirements. Following the delivering (ofexogenous materials), an additional incubation period may take place Dfor a duration according to the specification or requirements of theassay. The additional incubation period may be followed with collectingof samples 706. Additionally or alternatively, the step of deliveringmay be repeated, according to assay specification, in which eachdelivery may be followed with an additional incubation period.

The final step is performing analysis 708 to the samples collected.Additionally or alternatively, the embryo may be analyzed, includingimaging throughout the process or at specific time periods.

Identification & Delivering:

The classical assays for studying angiogenesis in vivo include the chickembryo chorioallantoic membrane (CAM). The chorioallantoic membrane(CAM) was first used to study tumor angiogenesis by grafting tumorsamples onto its surface on day 8 of incubation. Since then, thechorioallantoic membrane (CAM) assay has been used to identify almostall of the known angiogenic factors and to assess the angiostaticactivity of a variety of natural and synthetic compounds.

Reference is now made to FIG. 8, there is provided an illustrativeexample of different blood vessels branching, which is generallyindicated at 800, demonstrating responses around an implant, forexample, onto a chorioallantoic membrane (CAM), observed afterimplanting.

The illustrative example 800 includes partial sectional top view of thechorioallantoic membrane (CAM) 802, the transplant 804 and various bloodvessel such as: blood vessels with no branching 812; blood vessels withbranching around the transplant 814; and blood vessels with branchingfurther out of the transplant.

Reference is now made to the flowchart of FIG. 9A, there is provided apossible method, which is generally indicated at 900A, for use with theautomated system for the collection of biological samples. The methodincludes the steps:

In step 902A, obtaining an automated sample collection system such asdescribed hereinabove;

In step 904A, obtaining at least one fertilized egg;

In step 906A, optionally, incubating the fertilized egg for a firstduration period;

In step 908A, an applicator delivering exogenous material to thefertilized egg;

In step 910A, incubating the fertilized egg for an additional durationperiod; and

In step 912A, collecting at least one sample from the fertilized egg.

Where appropriate, step 908A may be further subdivided into the steps:

-   -   removing at least a part of the egg shell of the fertilized egg        thereby exposing at least part of the chorioallantoic membrane        (CAM); and    -   placing a population of cells in contact with the        chorioallantoic membrane (CAM).

It is noted that step 908A and step 910A may be repeated a plurality oftimes, for example, where a variety of types of exogenous material areto be applied. Thus, where required, a population of cells may beapplied to the chorioallantoic membrane (CAM) of a fertilized egg andthe fertilized egg then returned to the incubator for a period of timesufficient to allow engraftment of the implant to occur. The applicatormay then be used to deliver another exogenous material such as achemical or biological agent before again incubating the fertilized egg.

Where appropriate, the method may further include the steps of removinga part of the egg shell of the fertilized egg, or removing the whole eggfertilized shell, thereby exposing and providing access to thechorioallantoic membrane (CAM). The modulator may be administered usinga variety of techniques, such as topical administration, subcutaneousadministration, injection into the explant, injection into the explantvasculature, injection into fertilized egg vasculature and the like, aswell as combinations thereof.

It is further noted that, in order to improve engraftment in thefertilized egg, the fertilized egg immune response may be reduced byirradiating, using X-rays, gamma rays or the like, prior to engraftmentof the implanted cells. Such irradiation may inhibit the normalxenograft rejection of an intact host immune system. Furthermore, forengraftment of blood and bone marrow malignancies, irradiation may makehematopoietic niches available to absorb the donor cells in the hostbone marrow. It is noted that such irradiation typically leaves the egghost free of compounds that may interfere with the growth of the graftin the therapeutic conditions that are being tested. Additionally oralternatively, other immunosuppressant drugs such as cyclosporine or thelike may be used to improve engraftment due to immunosuppression of thehost rejection response, as well as the anti-apoptotic effect that maylimit graft cell death.

Reference is now made to the flowchart of FIG. 9B, there is providedanother possible method, which is generally indicated at 900B, for usewith the automated system for the collection of biological samples. Themethod includes the steps:

In step 902B, configuring an automated sample collection systemaccording to the desired assay;

In step 904B, pricking the chorioallantoic membrane (CAM) of the atleast one fertilized egg. Optionally, this step may be carried out by ashell removal apparatus such as described in FIG. 4B;

In step 906B, delivering at least one exogenous material to thefertilized egg, by an applicator. Where appropriate, the step furthercomprises various activities according to the assay specification suchas: engrafting, by the delivery mechanism (FIG. 2A, item 142), apopulation of cells to the chorioallantoic membrane (CAM); placing, bythe delivery mechanism (FIG. 2A, item 142), a population of cells incontact with the chorioallantoic membrane (CAM); and injecting, by thedelivery mechanism (FIG. 2A, item 142), exogenous material into adesignated blood vessel of chorioallantoic membrane (CAM) or into otherelements of the embryonated egg. A population of cells may also beapplied as a solid mass contained within a pre-formed gel “plug” (suchas matrigel, collagen, alginate or the like). A gel plug containing highlevels of pro-angiogenic agents may greatly improve the rate ofengraftment success, especially when smaller numbers of cells areincorporated in the plug.

In step 908B, incubating the fertilized egg for an additional durationperiod; and

In step 910B, harvesting at least one sample from the fertilized egg, bythe sample collector.

Where appropriate, step 902B may be further subdivided into the steps:

-   -   setting various configuration parameters associated with the        automated sampling system such as: a harvesting date and time;        an engrafting date and time; an injecting date and time; an        identified peak time for harvesting; a sample cell extracting        days and the like.

Where appropriate, step 904B may be further subdivided into the steps:

-   -   removing at least a part of the egg shell of the fertilized egg,        preferably at the widest side comprising the air pocket, thereby        exposing at least part of the chorioallantoic membrane (CAM);    -   optionally, puncturing the chorioallantoic membrane (CAM) to        enable the air pocket to shift to a desired location; and    -   optionally, placing a tube into the chorioallantoic membrane        (CAM). The placement of the tube may encourage blood vessels to        grow around the tube.

Where appropriate, step 910B may further include the steps:

-   -   In step 912B—analyzing the harvested sample;    -   In step 914B—analyzing the imaging captured during the process,        or captured at a given time/time duration;    -   In step 916B—analyzing the embryo.

It is noted that step 908B and step 910B may be repeated a plurality oftimes, for example, where a variety of types of exogenous material areto be applied. Thus, where required, a population of cells may beapplied to the chorioallantoic membrane (CAM) of a fertilized egg andthe fertilized egg then returned to the incubator for a period of timesufficient to allow engraftment of the implant to occur. The applicatormay then be used to deliver another exogenous material such as achemical or biological agent before again incubating the fertilized egg.

Reference is now made to the flowchart of FIG. 9C, there is provided apossible method of injecting, which is generally indicated at 900C, foruse with the automated system for the collection of biological samples.

As appropriate, directing the injection into the designated blood vesselin the direction of blood flow is required to make injection efficient.

Optionally, a flow-detector is used to determine the blood flowdirection of the designated blood vessel.

Optionally, an image processing technique is used and is configured todetermine the direction of blood flow according to the shape of thedesignated blood vessel.

Optionally, the system is operable to identify the umbilical veinconnecting the chorioallantoic membrane (CAM) and the embryo of theembryonated egg, and further determine that the direction of blood flowin the umbilical vein is from the chorioallantoic membrane (CAM) to theembryo.

Optionally, the system is operable to inject the exogenous materialeffectively by using a delivery mechanism that comprises a 30 gaugeneedle. Alternatively the gauge of the needle may be selected fromwithin the range 25-34, in still other embodiments, needles with gaugesof 24 and below may be preferred.

Optionally, the system is operable to inject the exogenous material andis configured to keep the delivery mechanism in the designated bloodvessel for 10 seconds.

Optionally, the system is operable to inject the exogenous material andis configured to apply surgical glue when the delivery mechanism iswithdrawn from the designated blood vessel. As appropriate, the jacketedneedle may be used such that the jacket releases the glue uponwithdrawal.

The method 900C includes the steps:

In step 902C, selecting a designated blood vessel having a knowndirection of blood flow;

In step 904C, determining the direction of the blood flow in thedesignated blood vessel; and

In step 906C, optionally using a flow-detector to determine the bloodflow direction in the designated blood vessel;

In step 908C, optionally using image processing technique configured todetermine the direction of blood flow in the designated blood vesselaccording to the shape of the designated blood vessel;

In step 910C, optionally an umbilical vein is identified connecting thechorioallantoic membrane (CAM) and the embryo; and further in step 912C,the flow direction of blood flow is determined to be from thechorioallantoic membrane (CAM) to the embryo.

In step 914C, directing the injection into the designated blood vesselin the direction of blood flow;

In step 916C, optionally using a delivery mechanism comprising a 30gauge needle, rather than 29 gauge needle;

In step 918C, optionally keeping the delivery mechanism in thedesignated blood vessel for 10 seconds;

In step 920C, optionally applying surgical glue when the deliverymechanism is withdrawn from the designated blood vessel;

It is noted that the glue may be inserted into a jacketed needle, forexample, before injecting, thus upon withdrawal of the deliverymechanism the glue is being released around the needle automatically.

Reference is now made to the flowchart of FIG. 9D, there is provided apossible method of engrafting, which is generally indicated at 900D, foruse with the automated system for the collection of biological samples.

As appropriate, following laboratory experiment, it has been exploredthat the junction between two blood vessels is more appropriate forengrafting. Thus, the process of engrafting the exogenous materialcomprises identifying a junction between a first blood vessel and asecond blood vessel on the chorioallantroic membrane (CAM), andengrafting the population of cells at the junction.

It is noted that the selection of the approriate junction for engraftingis not trivial and requires to know the blood vessel network of thechorioallantroic membrane (CAM) and other embryo associated organs toenable ‘picking’ the right location. Further, it is essential to know ifa blood vessel is a vein or an artery, which is associated with theinjection of the exogenous material, using an appropriate deliverymechanism, into the chorioallantroic membrane (CAM).

The method 900D includes the steps:

In step 902D, identifying a first blood vessel in the chorioallantoicmembrane (CAM) of the fertilized egg;

In step 904D, identifying a second blood vessel in the chorioallantoicmembrane (CAM) of the fertilized egg;

In step 906D, identifying a junction between the first blood vessel andthe second blood vessel;

In step 908D, engrafting a population of cells at the identifiedjunction of blood vessels;

In step 910D, setting the time period of incubation to be sufficient toallow engraftment of the population of cells to grow in thechorioallantoic membrane (CAM) of the fertilized egg; and

In step 912D, performing analysis of the grown population of cellsaccording to the specification of the designated assay associated withthe fertilized egg.

Reference is now made to the flowchart of FIG. 9E, there is provided apossible method of pricking, which is generally indicated at 900E, foruse with the automated system for the collection of biological samples.

Commonly, the automatic process of delivering is performed after theprocess of pricking, usually few days before. Alternatively, prickingmay be performed on the same day prior to delivering the exogenousmaterial.

Pricking the chorioallantroic membrane (CAM) of the at least onefertilized egg may be performed by the shell removal apparatus. Theprocess of pricking may include removing at least a part of the eggshell of at the widest side comprising the air pocket and exposing thechorioallantoic membrane (CAM) to allow puncturing the chorioallantoicmembrane (CAM) and further enable the air pocket to shift to a desiredlocation. Additionally, a tube may be placed into the chorioallantoicmembrane (CAM) to encourage blood vessels to grow around the tube and toease the actions of engrafting and injecting. The tube end may be coatedwith pro-angiogenic material, or release an air flow containing areduced or increased concentration of oxygen (15-30%). Alternatingapplication of diverse oxygen concentrations may result in robust bloodvessel growth, for example at E5 17% oxygen may induce hypoxia drivingblood vessel proliferation, while following implanting of exogenousmaterial, an increase in oxygenation may improve implanted cell vitalityand release of growth stimulatory signals.

Thus, the method 900E includes the steps:

In step 902E, removing at least a part of the shell of the fertilizedegg at the wide side which comprises the air pocket (FIG. 1A, item 70);

In step 904E, exposing at least a part of the chorioallantoic membrane(CAM) of the fertilized egg;

In step 906E, puncturing the chorioallantoic membrane (CAM) of thefertilized egg is performed to enable the air pocket to move to thehighest point wherever the shell is pricked; and

In step 908E, optionally a tube is placed into the chorioallantoicmembrane (CAM) such that a designated blood vessel may be encouraged togrow around the tube. This may take several days, thus it is suggestedto be applied at the time of pricking, few days before delivering.

It is further noted that the step of pricking is commonly performed on adifferent day before the delivering step (step 908A, FIG. 9A).Alternatively, it may be performed on the same day. Accordingly, whenthe chorioallantoic membrane (CAM) is punctured, some Amnium may beremoved.

Egg Sealing Unit:

Reference is now made to FIG. 10, in which there is provided anembodiment of an egg sealing unit, which is generally indicated at 1000,of the currently disclosed subject matter. The egg sealing unit diagram1000 comprises a sampler 1160, an imaging adaptor 1170, and anapplicator 1180.

The imaging adaptor 1170 may comprise a conduit 1172 for light travelingbetween the CAM and the egg shell surface, a ring 1174 and a seal 1176.

The conduit 1172 for light traveling between the CAM and the egg shellsurface, may be used for transmitting lightwaves (emitted from a bright,fluorescent or laser light source, for example) and optionally includesa series of optical modifiers such as lenses and filters. At the end ofthe light conduit in proximity to the CAM, the light conduit 1172 mayattach to a ring 1174 (plastic or silicone, for example) that is lyingon the CAM, such that its light beam is focused on the CAM andxenografted material present within the confines of the ring 1174. Atthe end of the light conduit 1172 in proximity to the egg shell surface,the light conduit 1172 may attach to an imaging unit 1170. The lightconduit 1172 is impermeable to water such that no condensation will formwithin, allowing constant unhampered imaging of the ring 1174 and itscontents on the CAM.

The light conduit 1172 can be made of glass, or any suitable lighttransmitting material. Multiple light conduits may be incorporated intoa single egg sealer unit 1000, such that multiple rings containingexogenous materials can be monitored within the same egg simultaneously.

The seal 1176 may comprise a gel, wax or other sealant sealing the eggshell opening to the light conduit 1172, such that no contamination canenter the egg, and that the interior of the egg does not dry out.

The ring 1174 may be placed on the CAM up to several days prior toimplantation of the exogenous material, during this period the ring 1174undergoes a form of binding with the CAM such that it cannot be movedwithout tearing the CAM, furthermore liquids and solids placed withinthe ring 1174 are confined to the ring area and do not leak under thering 1174. The ring 1174 whereabouts is therefore a suitable indicatorfor determining the location of the exogenous material on the CAM, forexample for placing the egg sealing unit 1000, or for directing theapplicator 1180 or sampler 1160 or imager 1170.

The sampler 1160 may be incorporated into the egg sealing unit 1000,comprising the apparatus described in the Sampler section.

The applicator 1180 may be incorporated into the egg sealing unit 1000,comprising the apparatus described in the Applicator section.

In one example, multiple rings may be placed on the CAM, each having adifferent identifier (colour, number or name, for example), eachcontaining a different exogenous material, such as cancer cells fromdifferent patients or sources. Thus, drug sensitive cells, tissues ororganisms and drug resistant cells, tissues or organisms can beengrafted in the same egg and treated under equivalent intra-eggconditions with the same external modulators.

EXAMPLES

For illustrative purposes only, a selection of examples are presentedbelow to demonstrate the usefulness of various embodiments of theautomated biological sample collection system 200A (FIG. 2A):

Growth of Viruses for Generation, Testing or Development of Vaccines

Viruses may be produced in embryonated chick eggs after injection intothe allantois, blood supply, yolk sac, or chorion. By using a biologicalsample collection system such as described hereinabove, the accuracy ofthe injection may be increased. This is particularly so when shell-lesseggs are incubated. Furthermore, it is noted that the use of shell-lesseggs also allows for rapid and accurate identification of dead embryoswhich may be readily removed.

Accordingly, eggs may be broken into individual compartments of the eggsupport apparatus, optionally this step may be performed by theshell-removal mechanism, although alternatively this step may be carriedout manually. The eggs may be incubated to the appropriate age for theparticular virus and administration route.

Once the embryos reach the appropriate age, the egg support apparatusmay move the eggs to the applicator where, using machine vision, theappropriate portion of the egg (allantois, amniotic sac, yolk sac, bloodvessel) may be identified, and injected robotically, for example, usingan automated version of a syringe-holding device such as that describedby Kelling and Schipper, 1976.

For example, the yolk or appropriate blood vessels may be identified bymeans of their color, shape, size or other chemical, physical orbiological characteristics, and the syringe may thereby be guidedtowards them automatically. Although the allantois and amniotic sac maybe more difficult to identify automatically, an automated algorithm maybe generated in order to allow identification of such elements.

Alternatively, an operator using a video-link to the inside of themachine, may perform the initial positioning of the manipulator, toallow the injection to proceed automatically thereafter.

While still in the chamber, dead embryos, or those sufficientlygrowth-stunted to be likely to die may be readily identified andremoved.

Assays of Chemotherapies on Human or Mammalian Cancer Cells Transplantedto the Egg

A) Hematological Malignancies:

i) Patient Samples of Blood Cancer for Theranostics.

According to another application, blood cancer samples may be collectedfrom individual patients for theranostics. A suspension of blood cancercells taken from the peripheral blood or bone marrow of patientssuffering from candidate conditions, such as leukemia, lymphoma, myelomaand the like, may be prepared for injecting into eggs.

Fertilized shell-less eggs may be prepared in individual compartments,either manually or using a shell removal apparatus. The shell-less eggsmay be incubated to the appropriate age for each type of virus andadministration route.

Upon the eggs reaching sufficient maturity, typically after 7-9 days ofincubation for chicken eggs or 8-11 days for turkey eggs, the tray ofthe egg support mechanism may be moved to the applicator.

Using machine vision, appropriate blood vessels in the eggs may beidentified, by color and size, for example. The manipulator may guide asyringe toward the blood vessels. The syringe would typically be filledwith the prepared suspension of blood cancer cells with typically about1-2 million cells being injected into each egg.

Following the application of cancerous cells, the tray of the eggsupport apparatus may be returned to the incubator for an additional 2-3days during which the cancer may become engrafted.

Following the engraftment of the cancer, the applicator may be used toinject chemotherapeutic agents intravenously.

Following a third incubation period, the bone marrow or other otherembryo tissues such as liver, spleen or the like, may be assayed, eithermanually or using the sampler, for the presence of human cancer cells.Variously, sampling may use the polymerase chain reaction (PCR) forhuman DNA, or human cell-surface markers by fluorescence aided cellsorting, or human cell death markers. Whole animal imaging may use cellsmade fluorescent in the infrared with lentiviral vectors drivingexpression of appropriate proteins such as Turbo635 (Evrogen).

ii) Blood Cell Lines for Drug Development.

Laboratory cancer cell lines may be obtained which are engineered toexpress far-red fluorescing proteins such as m-cherry, Turbo635(Evrogen) or the like.

As above, fertilized shell-less eggs may be prepared in individualcompartments, either manually or using a shell removal apparatus. Theshell-less eggs may be incubated to the appropriate age for each type ofvirus and administration route.

Upon the eggs reaching sufficient maturity, typically after 7-9 days ofincubation for chicken eggs or 8-11 days for turkey eggs, the tray of athe egg support mechanism may be moved to the applicator.

Using machine vision, appropriate blood vessels in the eggs may beidentified, by color and size, for example. The manipulator may guide asyringe toward the blood vessels. The syringe would typically be filledwith the cancer cell lines of blood cancer cells with typically about1-2 million cells being injected into each egg.

Following the application of cancerous cells, the tray of the eggsupport apparatus may be returned to the incubator for an additional 2-3days during which the cancer may become engrafted.

Following the engraftment of the cancer, the applicator may be used toinject agents, such as chemotherapeutic agents and the like,intravenously.

Following a third incubation period, the sampler may use whole-bodyimaging with green exciting light to assay the killing of cancer cellsby therapeutic agents.

Alternatively, non-engineered cells may be used, and cells tagged withmagnetic nanoparticles such that the sampler may use MRI imaging.

B) Solid Cancers

i) Patient Samples for Theranostics

Tumor tissue may be obtained from patients by removal during surgery orbiopsy, for example. Tissue may be cut into 1 mm cubes using a Mcilwaintissue chopper, for example. Alternatively, tissue may be minced andsuspended in a gel such as matrigel, and applied to the chorioallantoicmembrane (CAM).

The biological sampling system may be utilized to prepare suitable hosteggs, for example, using the methods described above.

The applicator may be used to place the tumor tissue cubes onto thechorioallantoic membrane (CAM) of E8 chicken embryos, for example. Theeggs may be incubated again for about three days until engraftment ofthe tissue occurs.

Following the engraftment of the cancer, the applicator may be used toinject chemotherapeutic agents intravenously. Lipid solublechemotherapeutic agents can be applied to the chorioallantoic membrane(CAM) without injection. Phototherapy activated-drugs (based onporphyrins) can also be used on CAM-grafted tumors. Typically, we haveto calibrate route of drug administration for each chemical—watersoluble drugs also can be dripped on the CAM. Where suitable, chemicalagents may be formulated such that they can be dripped or placed on theCAM distal to the implanted exogenous material, such that they areabsorbed by the underlying CAM blood vessels thereby entering the chickblood stream.

Following a third incubation period of, say, 3-5 days, grafted tumorpieces in treated and untreated embryos may be photographed, and theirsize compared. Thus candidate drugs may be compared for theireffectiveness in reducing tumor growth.

ii) Cell Lines for Drug Development.

Laboratory cancer cell lines may be obtained which are engineered toexpress green fluorescent protein, for example. A suspension may beprepared of such cells in a gelling matrix material (e.g. matrigel orpuramatrix).

The biological sampling system may be utilized to prepare suitable hosteggs, for example, using the methods described above.

The applicator may be used to place the cell suspension upon the CAM ofE8 chick embryos. The suspension may be contained upon the CAM within aplastic ring, for example. The eggs may be incubated again for aboutthree days until engraftment of the tissue occurs.

Following the engraftment of the cancer, the applicator may be used toinject chemotherapeutic agents intravenously.

Following a third incubation period of, say, 3-5 days or so, graftedtumor pieces of the laboratory line cancer cells in treated anduntreated embryos may be photographed, and their size compared. Thevitality or metabolic profile of the cancer cells may be rapidlyassessed through imaging of the reporter label (e.g., GFP fluorescence),or following tumor tissue processing, biomarkers can be analyzed (e.g.,by immunohistochemistry, Western blot, flow cytometry, PCR, whole mountimmunofluorescence. Thus candidate drugs may be compared for theireffectiveness in reducing tumor growth, as well as their ability toreach the tumor in detectable concentrations.

iii) Metastasis Studies

The biological sampling system may be used to detect metastasis ofapplied cancer cells to the internal organs of an embryo, usingwhole-animal imaging techniques such as MRI, whole animal fluorescenceimaging, or dissection and histological or FACS analysis, for example,as described above.

Assays for Angiogenic Properties of Substances Applied to theChorioallantoic Membrane (CAM)

Preparations of angiogenic or anti-angiogenic solutions may be absorbedonto 1 cm radius pieces of filter paper, for example. The applicator ofthe biological sampling system described herein may be used to placesuch preparations onto the chorioallantoic membrane (CAM) of E5 embryos,for example.

Following an incubation period of about 2-7 days, the sampler may beused to measure the generation or reduction of blood vessels, forexample, by using photography of the chorioallantoic membrane (CAM) andautomated image analysis.

Assays of Toxicity to the Embryo (Teratogenicity), Transplanted Human orMammalian Skin Transplanted to the CAM, Including Irritation,Sensitization etc.

i) Teratogenicity Testing

New compounds for use in industrial, medical, cosmetic or otherindustries would be injected intravascular as above, and embryos may beexamined at E18 or earlier for morphological abnormalities.

ii) Dermatological Testing

Human skin, consisting of epidermis and a few millimeters of dermis maybe obtained (i.e. from cosmetic surgery clinics), and cut into 6 mmrings with a dermal punch. The tissue may be placed dermis side down onthe chorioallantoic membrane (CAM) of E6-9 eggs, and further incubatedfor two days until engraftment. Optionally, the chorioallantoic membrane(CAM) can be mildly abraded with lens tissue to cause bleeding andthereby improve engraftment of the skin. The cutting of the skin and theapplication of the samples may be performed automatically by theapplicator or manually, as required.

Following engraftment, the skin may be treated with potential irritantsor sensitizers, UV irradiated, treated with suntan lotion etc. andincubated for an additional time.

Following further incubation, the skin may be assayed by histology etc,using conventional methods. It is noted that the sampler may furtherassay the skin implant during the incubation process, or at the end ofsuch process, using a non-invasive imaging system.

Additional dermatological applications may include testing for theintroduction of material such as: viruses (herpes), genes (genetherapy), examination of transcutaneous absorption (by taking bloodsamples from the embryo), bacteria into the skin (acne) or the like andcombinations thereof.

The scope of the disclosed subject matter is defined by the appendedclaims and includes both combinations and sub combinations of thevarious features described hereinabove as well as variations andmodifications thereof, which would occur to persons skilled in the artupon reading the foregoing description.

In the claims, the word “comprise”, and variations thereof such as“comprises”, “comprising” and the like indicate that the componentslisted are included, but not generally to the exclusion of othercomponents.

It should be appreciated to those skilled in the art that the inventionmay not be limited to the details of the foregoing illustrativeembodiments and that the present invention may be use various otherembodiments in other specific forms without departing from the nature oressential attributes thereof. The present embodiments are therefore tobe considered in all respects as illustrative and not restrictive.

Those skilled in the art to which this invention pertains will readilyappreciate that numerous changes, variations and modifications can bemade without departing from the scope of the invention mutatis mutandis.

Technical and scientific terms used herein should have the same meaningas commonly understood by one of ordinary skill in the art to which thedisclosure pertains. Nevertheless, it is expected that during the lifeof a patent maturing from this application many relevant systems andmethods will be developed. Accordingly, the scope of the terms such ascomputing unit, network, display, memory, server and the like areintended to include all such new technologies a priori.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to” and indicatethat the components listed are included, but not generally to theexclusion of other components. Such terms encompass the terms“consisting of” and “consisting essentially of”.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the composition or method.

As used herein, the singular form “a”, “an” and “the” may include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

The word “exemplary” is used herein to mean “serving as an example,instance or illustration”. Any embodiment described as “exemplary” isnot necessarily to be construed as preferred or advantageous over otherembodiments or to exclude the incorporation of features from otherembodiments.

The word “optionally” is used herein to mean “is provided in someembodiments and not provided in other embodiments”. Any particularembodiment of the disclosure may include a plurality of “optional”features unless such features conflict.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween. It should be understood,therefore, that the description in range format is merely forconvenience and brevity and should not be construed as an inflexiblelimitation on the scope of the disclosure. Accordingly, the descriptionof a range should be considered to have specifically disclosed all thepossible sub-ranges as well as individual numerical values within thatrange. For example, description of a range such as from 1 to 6 should beconsidered to have specifically disclosed sub-ranges such as from 1 to3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc.,as well as individual numbers within that range, for example, 1, 2, 3,4, 5, and 6 as well as non-integral intermediate values. This appliesregardless of the breadth of the range.

It is appreciated that certain features of the disclosure, which are,for clarity, described in the context of separate embodiments, may alsobe provided in combination in a single embodiment. Conversely, variousfeatures of the disclosure, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination or as suitable in any other describedembodiment of the disclosure. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Although the disclosure has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the disclosure.

The invention claimed is:
 1. A method for use in an automated samplecollection system for collecting a set of biological samples from atleast one fertilized egg, said system comprising: at least one incubatorconfigured to control the environment of said at least one fertilizedegg; a shell removal apparatus configured to remove at least a sectionof the shell providing access to a chorioallantroic membrane (CAM) ofsaid at least one fertilized egg; at least one applicator configured todeliver exogenous material to the fertilized egg; at least one samplerconfigured to collect samples from said at least one fertilized egg; andat least one controller unit, said method for operating said samplecollection system such that the associated process of collecting thesamples is performed in an improved manner, the method comprising thesteps of: configuring, by said controller unit, the automated samplecollection system according to at least one automated setup parameter;pricking, by said shell removal apparatus, the chorioallantroic membrane(CAM) of the at least one fertilized egg; delivering, by said at leastone applicator, at least one exogenous material to said at least onefertilized egg using at least one delivery mechanism associated withsaid at least one sampler; incubating, by said at least one incubator,the at least one fertilized egg for a period of incubation; harvesting,by said at least one sampler, at least one sample from the at least onefertilized egg, wherein the step of delivering at least one exogenousmaterial, comprises injecting, by said at least one delivery mechanism,the at least one exogenous material into a designated blood vessel ofsaid at least one fertilized egg; and directing the injection into saiddesignated blood vessel in the direction of blood flow.
 2. The method ofclaim 1, wherein the step of pricking the chorioallantroic membrane(CAM) of the at least one fertilized egg, comprises: removing at least apart of the shell of the at least one fertilized egg at the widest sidecomprising an air pocket; and exposing at least a part of thechorioallantoic membrane (CAM).
 3. The method of claim 2, wherein thestep of pricking, further comprises: placing a tube into thechorioallantoic membrane (CAM).
 4. The method of claim 1, wherein thestep of delivering at least one exogenous material comprises engrafting,by said at least one delivery mechanism, a population of cells to saidat least one fertilized egg.
 5. The method of claim 1, furthercomprising: determining the direction of blood flow in the designatedblood vessel.
 6. The method of claim 1, further comprising: selectingsaid designated blood vessel having a known direction of blood flow. 7.The method of claim 5, wherein the step of determining the direction ofblood flow in the designated blood vessel comprises: using aflow-detector to determine the blood flow direction of said designatedblood vessel.
 8. The method of claim 5, wherein the step of determiningthe direction of blood flow in the designated blood vessel, comprises:using image processing technique configured to determine the directionof blood flow according to the shape of said designated blood vessel. 9.The method of claim 1, wherein the step of directing the injection intosaid designated blood vessel in the direction of blood flow, comprises:identifying an umbilical vein connecting the chorioallantoic membrane(CAM) and an embryo of said at least one fertilized egg, and injectingsaid exogenous material into the umbilical vein in a direction from thechorioallantoic membrane (CAM) to the embryo.
 10. The method of claim 1,wherein said at least one exogenous material is selected from at leastone of a group consisting of: cells, tumor cells, bacteria, viruses,chemicals, drugs, skin explants and external modulators.
 11. The methodof claim 4, wherein the period of incubation is selected to besufficient to allow engraftment of said population of cells to thechorioallantoic membrane (CAM) of said at least one fertilized egg. 12.The method of claim 4, wherein the step of engrafting at least oneexogenous material comprises: identifying a junction between a firstblood vessel and a second blood vessel on said chorioallantroic membrane(CAM), and engrafting said population of cells at said junction.
 13. Themethod of claim 1, further comprising analyzing, by at least one imagingunit, the chorioallantoic membrane (CAM) and the embryo at apre-configured period of time.
 14. A method for use in an automatedsample collection system for collecting a set of biological samples fromat least one fertilized egg, said system comprising: at least oneincubator configured to control the environment of said at least onefertilized egg; a shell removal apparatus configured to remove at leasta section of the shell providing access to a chorioallantroic membrane(CAM) of said at least one fertilized egg; at least one applicatorconfigured to deliver exogenous material to the fertilized egg; at leastone sampler configured to collect samples from said at least onefertilized egg; and at least one controller unit, said method foroperating said sample collection system such that the associated processof collecting the samples is performed in an improved manner, the methodcomprising the steps of: configuring, by said controller unit, theautomated sample collection system according to at least one automatedsetup parameter; pricking, by said shell removal apparatus, thechorioallantroic membrane (CAM) of the at least one fertilized egg;delivering, by said at least one applicator, at least one exogenousmaterial to said at least one fertilized egg using at least one deliverymechanism associated with said at least one sampler; incubating, by saidat least one incubator, the at least one fertilized egg for a period ofincubation; harvesting, by said at least one sampler, at least onesample from the at least one fertilized egg; analyzing, by at least oneimaging unit, the chorioallantoic membrane (CAM) and the embryo at apre-configured period of time by using, by said at least one imagingunit, a non-invasive photographic technique selected from a groupconsisting of an imaging fluorescence-based photography technique; animaging luminescence-based photography technique; an imagingconventional illumination-based photography technique; an imagingultrasonic-based technique; an imaging X-ray based technique; an heatdetector based technique; radioactivity detector based technique; MRIbased technique; and combinations thereof.
 15. The method of claim 14,further comprising: using a high-resolution imaging fluorescence-basedphotography technique; marking each cell to be individuallyidentifiable; and indicating an associated cell type by expressingadditional fluorescent labels.
 16. The method of claim 14, wherein thestep of analyzing comprises testing, by said at least one imaging unit,the convergence of blood vessels towards the graft.
 17. The method ofclaim 14, wherein the step of analyzing comprises: testing, by said atleast one imaging unit, for variations in the distribution of density ofchorioallantoic membrane (CAM) blood vessels next to the site of thegraft.
 18. The method of claim 14, wherein the step of analyzing,comprises: mapping, by said at least one imaging unit, of blood vesselsbranching.
 19. The method of claim 1, further comprising monitoring atleast one activity selected from the group consisting of: the absorptionof the bolus; blood leakage from the blood vessels; bolus ejected fromthe at least one fertilized egg; and combinations thereof.